The immunosuppressant sanglifehrin A (SFA) is a mixed polyketide-peptide natural product from Streptomyces sp. A92-308110, also known as Streptomyces flaveolus or Streptomyces sp. DSM 9954—these titles are all used interchangeably in this and related documents (Sanglier et al., 1999; Fehr et al., 1999; WO 97/02285). Isolation of more than twenty structural analogues of sanglifehrin have been published to date, and SFA has one of the highest immunosuppressant activities among these analogues (Kallen et al., 2005; Sanglier et al., 1999). In SFA, the 22-membered macrolide backbone consists structurally of a polyketide carbon chain and a tripeptide chain. The peptide chain comprises one natural amino acid: valine, and two non-natural amino acids: (S)-m-tyrosine and (S)-piperazic acid, linked by an amide bond, and it is the β-nitrogen atom at position 1 of piperazic acid that is involved in amide bond formation, which stands in contrast to all other piperazic acid containing natural products isolated so far. In addition, a spirocyclic unit is linked to the macrolide by a polyketide long chain, forming a basket structure. The spirocyclic moiety contains nine chiral centers (SFA has seventeen in total) with a quaternary carbon center in the middle, which is unique in currently described natural products. A series of analogues have been directly isolated from fermentation broths of Streptomyces sp. A92-308110 (S. flaveolus), including sanglifehrin B, C, D, E, F, G, H, I, J, K and L (Fehr et al., 1999, Sanglier et al., 1999, WO98/07743). Sanglifehrin B (SFB), in particular, has been shown to possess higher immunosuppressive activity than SFA in MLR assays (Sanglier et al., 1999). Though the total synthesis of SFA and its macrocyclic analogues were achieved with heroic efforts (Sedrani et al., 2003; Nicolau et al., 1999; Paquette et al., 2002; Metternich et al., 1999), no specific in vivo and in vitro studies have been carried out on its biosynthetic pathway.
SFA has strong immunosuppressive activity (Powell and Zheng, 2006), inhibits HIV and HCV infection (Zander et al., 2003; Sokolskaja et al., 2004; Watashi et al., 2005) and prevents severe cardiac cell death caused by the pathological opening of mitochondrial membrane permeability transition pore (MPTP; Clarke et al., 2002). Compared with the immunosuppressants currently in clinical use, such as cyclosporin A (CsA), FK506 and rapamycin, SFA has a similar functional mechanism while having a different target effector site (Hartel et al 2006, Zhang & Liu., 2001; Zenke et al., 2001). CsA binds to cyclophilin A (CypA) (Handschumacher et al., 1984), whilst FK506 and rapamycin bind to FKBP, to form complexes (Schreiber, 1991); the CsA-CypA and FK506-FKBP complexes interact with the same target protein, calcineurin, thereby inhibiting the serine/threonine phosphatase activity of calcineurin, and blocking the production of cytokines, especially the transcription of interleukin 2 (IL-2), which finally lead to T cell arrest in G0-G1 stage (Liu et al., 1991). Rap-FKBP complexes interact with the protein kinase FRAP (also known as RAFT or mTOR) (Brown et al., 1994), and prevent phosphorylation of the IL-2 receptor on T cells, leading to arrest of the growing of T cells in G1-S stage. Whilst SFA has been shown to bind to cyclophilins such as Cyclophilin A and B, and inhibit their isomerase activities (Zenke et al 2001), currently the effector protein for SFA-CypA complex remains unknown.
Since the effector protein of the SFA-CypA complex has not yet been found, it was suggested that the immunosuppressive activity of SFA is not mediated directly via the the SFA-CypA complex. In the past 3 years, studies from many scientific groups have shown that SFA can competitively prevent NF-κB from binding the transcription site upstream of the P53 gene, to activate P53 and further inhibit the downstream Cyclin E-cdk2 phosphorylation of the signal pathway, thereby inhibiting the high phosphorylation of Rb in response to IL-2, and making cells insensitive to to IL-2, which forces them to remain in the G1-S stage (Zhang & Liu, 2001). Secondly, by an unknown mechanism, SFA inhibits production of IL-12p70 while not affecting the growth of human dendritic cells (Steinschulte et al., 2003). IL-12p70 plays a key role in regulating proliferation of Th1 and NK cells, and is the bridge linking innate immunity with adaptive immunity. In addition, the immunosuppressive drugs that are commercially available can lead to severe renal and central nervous system toxicity side effects (Paquette et al., 2002), thus their uses in some immune dysfunction diseases are hindered (for example, calcineurin is the underlying cause of both immunosuppressive and toxic effects of CsA and FK506). With the aim of developing an alternative immunosuppressant or immune modifier, other groups have carried out some development of SFA as a new generation of potent immunosuppressant with lower toxicity (WO 97/02285).
The study of structure-activity relationships between SFA macrocyclic fragments and CypA by X-ray diffraction showed that the tripeptide structure is embedded in the groove of CypA and is important for binding, while the side chain hydroxy group and carbonyl group respectively at positions 17 and 14 are not critical for binding; removal of the trans-diene from the saturated region C18-C22 reduces the binding constant 7 fold, suggesting the trans-diene stabilizes the conformation (Sedrani et al., 2003). A computer-modeling study shows that the spirocyclic unit of SFA may also contribute to the stability of the SFA-CypA binding (Pemberton et al., 2003). The crystal structure of the complete SFA-CypA complex shows that the binding regions in SFA-CypA are substantially the same as those in CsA-CypA, and both SFA and CsA mainly interact with residue W121, R55, H126, N102 and Q63; the C24-C32 chain between the macrocycle and the spirocyclic moiety make van der Waals contacts with residues I57, T119 and W121 of CypA; in addition, the presence of the long polyketide chain of SFA imposes a side-chain reorientation on W121, as compared with the crystal structure of CypA; within the spirocycle, only the methyl group C45 makes vdW contacts with side-chain atoms from I57 and F60 of CypA (Kallen et al., 2005).
The SFA-CypA complex can exist in a stable dimeric form, as shown by gel filtration chromatography. Based on crystal analysis, with the exception of the spirobicyclic and α-ketobutyrate moieties, all of the remaining parts of SFA are deeply buried in the dimer; the E,E-diene region C18-C22 is not involved in direct contacts with the CypA but instead forms vdW contacts with the meta-tyrosine of neighboring SFA within the dimer, which favors the dimeric association in the complex; the two SFA molecules make vdW contacts with each other in the region C18-C22; and a direct hydrogen bond links W121 of one CypA molecule and R148 of another CypA molecule in the dimer complex.
Using the streptomycete that is known to produce SFA, Streptomyces sp. A92-308110 (S. flaveolus), the inventors of the present invention cloned the biosynthetic gene cluster thereof, and further studied the biosynthesis of SFA by methods combining microbiology, molecular biology, biochemistry and organic chemistry. Through study of the biosynthesis, the enzymatic mechanism which generates distinctive chemical structures such as piperazic acid was revealed. Based on this, genetic modifications were made to the SFA biosynthetic pathway, and novel compounds were produced.
The present invention is particularly useful as it should enable the commercial application of recombinant DNA technology and biosynthetic engineering to increase the yield of sanglifehrins and generation of novel sanglifehrin analogues.